A simple and rapid DNA extraction method from whole blood for highly sensitive detection and quantitation of HIV-1 pro-viral

DNA by real-time PCR (Journal of Virological Methods 214 (2015) 37–42 )Authors :

Sally M. McFalla, Robin L. Wagnera, Sujit R. Jangama, Douglas H. Yamadaa, Diana Hardied, David M. KelsoPublished on :12 February 2015

Presented by :Manish ThakurM.Sc-1Roll No-6Center for Systems Biology & Bioinformatics

Aim

A huge amount of new cases of HIV-1 infection detected in infants.

WHO recommendations Infants should be tested at 4-6 week of age.

Virological test in infants is challenging because Low amount of maternal antibodies in infants.

PCR based methods for detection of pro-viral HIV-1 DNA in infants is most promising method.

Barriers in PCR based detection methods are Implementation of PCR testing protocols in resource-limited areas. DNA extraction is time consuming method Skilled person are required. Reagents & equipments are costly

Urgent need to improve access to early infant diagnosis particularly at peripheral sites where, increasingly, HIV clinical management is being provided.

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Introduction

For over come this problem dried blood spot (DBS) protocol is used.

Their is problem of Sample acquisition and transportation. Time consuming

Therefore a early diagnostic test is required. The use of filtration isolation of nucleic acids (FINA) as a viable method for point-of-care extraction because• Very sensitive and specific• Time saver The pro-viral copy number in each of these clinical samples was

calculated by comparison of the Cq of each sample to a standard curve run simultaneously.

DBS at

site of

treatmen

t.

Transported for testing

Return

back the results

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Material

Reference strains• 8E5-LAV cells frozen cell pellets of

4000cell/𝛍l• Dilute cell pellets for creating

standard curve

Clinical samples

• 100 𝛍l whole blood is taken from the infants less than 1year of age.

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Methods

DNA isolation

qPCR amplification and detection

Exogenous internal control

Statistical analysis

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DNA Isolation by FINA…..

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qPCR amplification and detection….

ZO5 polymerase was substituted for Tth

polymerase

The reverse transcription step was omittedas and

the qPCR reac-tions were run on the Stratagene

Mx3005p using the FAM and Cy5 channels for

data collection

HIV-1 amplicon was detectedby a 5fluorescein (FAM) conjugated probe

Internal control amplicon was detected by a

5conjugated Quasar670 probe

Number of modifications to qPCR reagents & protocol were made to adapt the assay for eventual incorporation into the FINA POC instrument under development

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Exogenous internal control

The 136-bp amplicon derived from the hydroxypyruvate reductase (HPR)gene from pumpkin plant was cloned into pZErOTM-2

Plasmids containing HPR target were spiked into PCR master mix containing the primers and probes for HPR gene at 500 copies per reaction..

Standard curves of 8E5-LAV cells were run with and without primers, probe and HPR plasmid to demonstrate consistency of HIV assay.

NegativePositive

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Statistical analysis…..

Cqvalues were plotted against log

(copy number) using Strata-gene’s MxPro Software to obtain standard curves. Slope parameters were estimated by linear regression of

Cqvalues vs log (copynumber).

For the slope estimates, 95%

Confidence Intervals were obtained using

GraphPad Prism v 6.0 . The value of the

slope was used to calculate PCR effi-

ciency.

To determine if the observed detection of proviral DNA in this single copy

study was significantly different from the predicted number of samples

with ≥1 acopy according to Poisson distribution, the two-

tailed P value was calculated using Chi-

square analysis using GraphPad

QuickCalcs

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Results

• Introduction of internal control

Standard curves of Cq v alues calculated from amplification plot versus log copy number of 8e5/LAV cells per 100 𝛍l whole blood.

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Graph (1) Raw data amplification plots of no template control (solid line; NTC) and a weak positive (dotted line; well C3).

• Determination of limits of detection and quantitation Improvements

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Graph (2) NTC straight line fit analysis. raw data{ -------- }, straight line{ _______ } fit from cycles 15–29 ,{ ______ } deviation from straight line fit;

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Graph (3) Weak positive well C3 straight line fit analysis. raw data{------}, straight line { ______ } fit from cycles 15–29 , { _______ }deviation from straight line fit.

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Pro-viral Copy No./100 𝛍l WB

No. of Positives % Positives (Cumulative)

<=10 2 7

11-25 3 17

26-50 7 41

51-100 8 69

101-200 5 86

201-300 3 97

>1000 1 100

• Evaluation of FINA performance with clinical infant specimens

Summary of quantitation of pro-viral DNA copy numbers from 29 HIV-1 positive infant specimens. The copy number was calculated from a standard curve of Cq values versus log copy number of 4000, 400, 40 and 10 8e5/LAV cells per 100 𝛍l whole blood in triplicate.

With in 61 specimen, one specimen did not give a valid amplification and was excluded from the analysis. 29 true positives and 31 true negatives were detected yielding 100% sensitivity and 100% specificity .

Conclusion…..

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An assay for the detection and quantitation of HIV-1 pro-viral DNA in whole blood specimens

Comprehensive Simple to use Highly sensitive

FINA extraction

Real-Time PCR

Under development for use in limited resource settings In this study, the introduction of an internal control and modifications to the FINA process are reported which greatly improve the analytical sensitivity.

The prototype used a larger DNA capture membrane diameter (11 mm) which required a much larger qPCR reaction volume (468 𝛍l) leading to reduced qPCR sensitivity and increased reagent cost.

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Future prospective….. Other assays such as the COBAS® Ampliprep/COBAS®Taq-man System amplify both HIV-1 viral RNA and pro-viral DNA for enhanced sensitivity. Efforts to improve the sensitivity of the FINA assay by amplifying viral RNA in addition to pro-viral DNA were not successful.

The Fusion 5 membrane used in the FINA module may capture the genomic DNA by entangling the DNA on the filter as demonstrated by an electron-microscopy image of the Whatman FTATM card with entangled DNA.

Detection and quantitation of pro-viral DNA is becoming an increasingly important biomarker for HIV-1 disease monitoring. Patients receiving highly active antiretroviral treatment typically achieve viral suppression rapidly.

The FINA extraction could be adapted for use with other inputs besides peripheral blood to look for reservoirs of HIV-1 infected cells.

HIV pro-viral testing could also be used to screen vaccine trial subjects for true infection.

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Thank You…….

Questions are welcome……